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Dynamics and hydration explain failed functional transformation in dehalogenase design

Nature Chemical Biology volume 10, pages 428–430 (2014)Cite this article

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Abstract

We emphasize the importance of dynamics and hydration for enzymatic catalysis and protein design by transplanting the active site from a haloalkane dehalogenase with high enantioselectivity to nonselective dehalogenase. Protein crystallography confirms that the active site geometry of the redesigned dehalogenase matches that of the target, but its enantioselectivity remains low. Time-dependent fluorescence shifts and computer simulations revealed that dynamics and hydration at the tunnel mouth differ substantially between the redesigned and target dehalogenase.

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Figure 1: Transplantation of the active site and the access tunnel from DbjA to DhaA.
Figure 2: Structural and biochemical properties of wild types and variants.
Figure 3: Dynamics and hydration of wild type and variants.

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NCBI Reference Sequence

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References

  1. Damborsky, J. & Brezovsky, J. Curr. Opin. Chem. Biol. 19, 8–16 (2014).

    Article  CAS  Google Scholar 

  2. Koehl, P. & Levitt, M. J. Mol. Biol. 293, 1161–1181 (1999).

    Article  CAS  Google Scholar 

  3. Siegel, J.B. et al. Science 329, 309–313 (2010).

    Article  CAS  Google Scholar 

  4. Röthlisberger, D. et al. Nature 453, 190–195 (2008).

    Article  Google Scholar 

  5. Jiang, L. et al. Science 319, 1387–1391 (2008).

    Article  CAS  Google Scholar 

  6. Kries, H., Blomberg, R. & Hilvert, D. Curr. Opin. Chem. Biol. 17, 221–228 (2013).

    Article  CAS  Google Scholar 

  7. Khersonsky, O. et al. Proc. Natl. Acad. Sci. USA 109, 10358–10363 (2012).

    Article  CAS  Google Scholar 

  8. Khare, S.D. et al. Nat. Chem. Biol. 8, 294–300 (2012).

    Article  CAS  Google Scholar 

  9. Giger, L. et al. Nat. Chem. Biol. 9, 494–498 (2013).

    Article  CAS  Google Scholar 

  10. Blomberg, R. et al. Nature 503, 418–421 (2013).

    Article  CAS  Google Scholar 

  11. Sato, Y. et al. Appl. Environ. Microbiol. 71, 4372–4379 (2005).

    Article  CAS  Google Scholar 

  12. Kulakova, A.N., Larkin, M.J. & Kulakov, L.A. Microbiology 143, 109–115 (1997).

    Article  CAS  Google Scholar 

  13. Chovancová, E., Kosinski, J., Bujnicki, J.M. & Damborsky, J. Proteins 67, 305–316 (2007).

    Article  Google Scholar 

  14. Prokop, Z. et al. Angew. Chem. Int. Edn Engl. 49, 6111–6115 (2010).

    Article  CAS  Google Scholar 

  15. Eisenmesser, E.Z. et al. Nature 438, 117–121 (2005).

    Article  CAS  Google Scholar 

  16. Henzler-Wildman, K. & Kern, D. Nature 450, 964–972 (2007).

    Article  CAS  Google Scholar 

  17. Bhabha, G. et al. Science 332, 234–238 (2011).

    Article  CAS  Google Scholar 

  18. Jimenez, R., Fleming, G.R., Kumar, P.V. & Maroncelli, M. Nature 369, 471–473 (1994).

    Article  CAS  Google Scholar 

  19. Halle, B. & Nilsson, L. J. Phys. Chem. B 113, 8210–8213 (2009).

    Article  CAS  Google Scholar 

  20. Jesenská, A. et al. J. Am. Chem. Soc. 131, 494–501 (2009).

    Article  Google Scholar 

  21. Chang, C.-W. et al. Proc. Natl. Acad. Sci. USA 107, 2914–2919 (2010).

    Article  CAS  Google Scholar 

  22. Jurkiewicz, P., Cwiklik, L., Jungwirth, P. & Hof, M. Biochimie 94, 26–32 (2012).

    Article  CAS  Google Scholar 

  23. Nilsson, L. & Halle, B. Proc. Natl. Acad. Sci. USA 102, 13867–13872 (2005).

    Article  CAS  Google Scholar 

  24. Baker, D. Protein Sci. 19, 1817–1819 (2010).

    Article  CAS  Google Scholar 

  25. Chovancova, E. et al. PLoS Comput. Biol. 8, e1002708 (2012).

    Article  CAS  Google Scholar 

  26. Sambrook, J. & Russell, D.W. Molecular Cloning: a Laboratory Manual. (Cold Spring Harbor Laboratory Press, New York, 2001).

  27. Iwasaki, I., Utsumi, S. & Ozawa, T. Bull. Chem. Soc. Jpn. 25, 226 (1952).

    Article  CAS  Google Scholar 

  28. Furse, K.E. & Corcelli, S.A. J. Phys. Chem. Lett. 1, 1813–1820 (2010).

    Article  CAS  Google Scholar 

  29. Ducruix, A. & Giegé, R. Crystallization of Nucleic Acids and Proteins: a Practical Approach. (Oxford University Press, New York, 1999).

  30. Teng, T.Y. J. Appl. Crystallogr. 23, 387–391 (1990).

    Article  CAS  Google Scholar 

  31. Vagin, A. & Teplyakov, A. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  32. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  33. Sheldrick, G.M. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Baker (University of Washington) for helpful comments on the manuscript. This work was supported by the European Regional Development Fund (CZ.1.05/1.1.00/02.0123), the Czech Ministry of Education (LO1214), the Czech Science Foundation (P208/12/G016 to J.S. and M.H. and P503/12/0572 to J.D.) and the 'Employment of Best Young Scientists for International Cooperation Empowerment' program (CZ1.07/2.3.00/30.0037 to J.B.), financed by both the European Social Fund and the state budget of the Czech Republic. The Academy of Sciences is acknowledged for the Praemium Academie award (M.H.). CERIT Scientific Cloud is acknowledged for providing access to their computing facilities under the Center CERIT Scientific Cloud program (CZ.1.05/3.2.00/08.0144).

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Author notes

  1. Jan Sykora, Jan Brezovsky and Tana Koudelakova: These authors contributed equally to this work.

Authors and Affiliations

  1. J. Heyrovsky Institute of Physical Chemistry of the ASCR, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Jan Sykora, Tatsiana Chernovets & Martin Hof

  2. Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment (RECETOX), Loschmidt Laboratories, Faculty of Science, Masaryk University, Brno, Czech Republic

    Jan Brezovsky, Tana Koudelakova, Andrea Fortova, Radka Chaloupkova, Veronika Stepankova, Zbynek Prokop & Jiri Damborsky

  3. Faculty of Science, University of South Bohemia in Ceske Budejovice, Ceske Budejovice, Czech Republic

    Maryna Lahoda & Ivana Kuta Smatanova

  4. Institute of Nanobiology and Structural Biology ASCR, v.v.i., Nove Hrady, Czech Republic

    Maryna Lahoda & Ivana Kuta Smatanova

  5. International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic

    Zbynek Prokop & Jiri Damborsky

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  1. Jan Sykora
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Contributions

J.S. and T.C. conducted fluorescence spectroscopy measurements. J.B. performed molecular modeling. T.K. and A.F. constructed the mutants. T.K. and Z.P. biochemically characterized the mutants. T.K., V.S. and R.C. conducted CD spectroscopy measurements. M.L. and I.K.S. determined the crystal structure. M.H. and J.D. conceived and supervised the project. J.S., J.B., T.K., M.H. and J.D. wrote the paper together.

Corresponding authors

Correspondence to Martin Hof or Jiri Damborsky.

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The authors declare no competing financial interests.

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Supplementary Note, Supplementary Results, Supplementary Figures 1–11 and Supplementary Tables 1–9. (PDF 2175 kb)

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Sykora, J., Brezovsky, J., Koudelakova, T. et al. Dynamics and hydration explain failed functional transformation in dehalogenase design. Nat Chem Biol 10, 428–430 (2014). https://doi.org/10.1038/nchembio.1502

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